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Abstract

Na,K-ATPase mediates net electrogenic transport by extruding three Na⁺ ions and importing two K⁺ ions across the plasma membrane during each reaction cycle. We mutated putative cation coordinating amino acids in transmembrane hairpin M5-M6 of rat Na,K-ATPase: Asp⁷⁷⁶ (Gln, Asp, Ala), Glu⁷⁷⁹ (Asp, Gln, Ala), Asp⁸⁰⁴ (Glu, Asn, Ala), and Asp⁸⁰⁸ (Glu, Asn, Ala). Electrogenic cation transport properties of these 12 mutants were analyzed in two-electrode voltage-clamp experiments on Xenopus laevis oocytes by measuring the voltage dependence of K⁺-stimulated stationary currents and pre-steady-state currents under electrogenic Na⁺/Na⁺ exchange conditions. Whereas mutants D804N, D804A, and D808A hardly showed any Na⁺/K⁺ pump currents, the other constructs could be classified according to the [K⁺] and voltage dependence of their stationary currents; mutants N776A and E779Q behaved similarly to the wild-type enzyme. Mutants E779D, E779A, D808E, and D808N had in common a decreased apparent affinity for extracellular K⁺. Mutants N776Q, N776D, and D804E showed large deviations from the wild-type behavior; the currents generated by mutant N776D showed weaker voltage dependence, and the current-voltage curves of mutants N776Q and D804E exhibited a negative slope. The apparent rate constants determined from transient Na⁺/Na⁺ exchange currents are rather voltage-independent and at potentials above -60 mV faster than the wild type. Thus, the characteristic voltage-dependent increase of the rate constants at hyperpolarizing potentials is almost absent in these mutants. Accordingly, dislocating the carboxamide or carboxyl group of Asn⁷⁷⁶ and Asp⁸⁰⁴, respectively, decreases the extracellular Na⁺ affinity.